SE466403B - SAID TO SYNTHETIZE Oligosaccharides - Google Patents

SAID TO SYNTHETIZE Oligosaccharides

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Publication number
SE466403B
SE466403B SE8801080A SE8801080A SE466403B SE 466403 B SE466403 B SE 466403B SE 8801080 A SE8801080 A SE 8801080A SE 8801080 A SE8801080 A SE 8801080A SE 466403 B SE466403 B SE 466403B
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substance
synthesis
oligosaccharide
process according
udp
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SE8801080A
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Swedish (sv)
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SE8801080D0 (en
SE8801080L (en
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Kurt G I Nilsson
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Kurt G I Nilsson
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Priority to SE8801080A priority Critical patent/SE466403B/en
Publication of SE8801080D0 publication Critical patent/SE8801080D0/en
Priority to DE68923177T priority patent/DE68923177T2/en
Priority to US07/603,699 priority patent/US5246840A/en
Priority to PCT/SE1989/000151 priority patent/WO1989009275A1/en
Priority to EP89904653A priority patent/EP0432157B1/en
Publication of SE8801080L publication Critical patent/SE8801080L/en
Publication of SE466403B publication Critical patent/SE466403B/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/26—Preparation of nitrogen-containing carbohydrates

Description

466 405 i Naturens egna katalysatorer, enzymer, uppvisar många attraktiva egenskaper, som t.ex. ofta absolut stereospecificitet, hög regio- och substratselektivitet samt hög katalytisk effektivitet vid milda betingelser. Man hyser därför idag förhoppningar om att kunna utnyttja enzymer för storskalig selektiv syntes av oligosackarider med färre syntessteg och därmed högre totalutbyten än med organisk-kemisk metodik. 466 405 in Nature's own catalysts, enzymes, exhibit many attractive properties, such as often absolute stereospecificity, high region and substrate selectivity and high catalytic efficiency under mild conditions. Therefore, there are hopes today of being able to use enzymes for large-scale selective synthesis of oligosaccharides with fewer synthesis steps and thus higher overall yields than with organic-chemical methodology.

Både hydrolaser (glykosidaser, EC klass 3.2) och glykosyltransferaser (EC klass 2.4) kan användas för syntes (glykosidaser: se Nisizawa et al, i "The Carbohydrates, Chemistry and Biochemistry, 2nd edition, vol.IIA, s.242-90 Academic Press, New York (1970)). Med glykosidaser används ofta omvänd hydro-1 lys (jämviktsreaktion) eller transglykosylering (kinetisk reaktion)för att åstadkomma syntes (se t.ex. K.G.I. Nilsson, Carbohydr. Res., vol. 167, s. 95-103 (1987).Med transferaser används nukleotidsocker (UDP-Gal, CMP-Sia, UDP-GalNAc, GDP-Fuc etc), vilka är relativt dyra, som donator för att åstadkomma syntes. Båda enzymtyperna har sina fördelar: Glykosidaser är ofta lättillgängliga och kan ibland användas direkt i en fermentationslösning, glykosyltransferaser uppvisar hög regio- och acceptorselektivitet. Använda var för sig uppvisar emellertid båda typerna av enzym betydande nackdelar för syntes av högre oligosacharider. Glykosidaser uppvisar ofta en icke-absolut regioselektivitet varför flera isomera produkter kan erhållas och produkt- uppreningen försvåras. Detta gör att glykosidaser ofta ej lämpar sig för syntes av högre oligosaccharider. Glykosyltransferaser förekommer i regel i låga koncentrationer i levande celler och är således ofta svårtillgängliga.Both hydrolases (glycosidases, EC class 3.2) and glycosyltransferases (EC class 2.4) can be used for synthesis (glycosidases: see Nisizawa et al, in "The Carbohydrates, Chemistry and Biochemistry, 2nd edition, vol.IIA, pp.242-90 Academic Press, New York (1970)) With glycosidases, reverse hydrolysis (equilibrium reaction) or transglycosylation (kinetic reaction) is often used to effect synthesis (see, e.g., KGI Nilsson, Carbohydr. Res., Vol. 167, p. 95-103 (1987). With transferases, nucleotide sugars (UDP-Gal, CMP-Sia, UDP-GalNAc, GDP-Fuc, etc.) are used, which are relatively expensive, as a donor to effect synthesis.Both types of enzymes have their advantages: Glycosidases are often readily available and can sometimes be used directly in a fermentation solution, glycosyltransferases show high regio- and acceptor selectivity.However, used separately, both types of enzyme show significant disadvantages for the synthesis of higher oligosaccharides.Glycosidases often show a non-absolute regioselectivity why several isomeric products can be obtained and product purification is made more difficult. This means that glycosidases are often not suitable for the synthesis of higher oligosaccharides. Glycosyltransferases usually occur in low concentrations in living cells and are thus often difficult to obtain.

Dessutom är glykosyltransferaserna beroende av kofaktorer (se ovant.In addition, glycosyltransferases are dependent on cofactors (see above.

Föreliggande uppfinning syftar till att utnyttja glykosidasers och glykosyltransferasers egenskaper så att effektiv synthes av oligosacharider uppnås. Detta erhålls enligt uppfinningen genom att glykosidas-katalyserad syntes av oligosacharidföreniäg?5åâeëf§kosyltransferaskatalyserad syntes av den högre oligosacharidföreningen. Lättillgängligt glykosidas används således för syntes av kortare oligosacharidföreningen, vars syntes inte ställer så stora krav på hög regioselektivitet hos enzymet som syntesen av den högre oligosachariden kräver och där således enligt uppfinningen ett glykosyltransferas utnyttjas.The present invention aims to utilize the properties of glycosidases and glycosyltransferases so that efficient synthesis of oligosaccharides is achieved. This is obtained according to the invention by glycosidase-catalyzed synthesis of oligosaccharide compound and cryosyltransferase-catalyzed synthesis of the higher oligosaccharide compound. Thus readily available glycosidase is used for the synthesis of the shorter oligosaccharide compound, the synthesis of which does not place as high demands on the high regioselectivity of the enzyme as the synthesis of the higher oligosaccharide requires and thus according to the invention a glycosyltransferase is used.

Principen illustreras av nedanstående reaktionsschema som inte är avsett att begränsa uppfinningens omfattning: glykosidas D-l H1 + ÅRZ -"'_"__*fi- D-IÅRZ + H1 ND2 D N glykosyltransferas D2 1AR2 + 3 466 403 I schemat på föregående sida symboliserar D1R1 glykosyldonator (oligosackarid, glykosid) med F-glykosid av en di- eller en högre oligosackarid, ND2 är en lämplig socker. nukleotid (CMP-Neu5Ac, UDP-Gal, UDP-GalNAc, GDP-Fuc, etc) och D2D1AR2 är den slutliga oligosacharidföreningen. Glykosidasreaktion kan också utföras som jämviktsreaktion. Mer än ett glykosidas och/eller transferas kan användas för syntes av högre oligosackarider.The principle is illustrated by the following reaction scheme which is not intended to limit the scope of the invention: glycosidase D1 H1 + ÅRZ - "'_" __ * fi- D-IÅRZ + H1 ND2 DN glycosyltransferase D2 1AR2 + 3,466,403 (oligosaccharide, glycoside) with F-glycoside of a di- or a higher oligosaccharide, ND2 is a suitable sugar. nucleotide (CMP-Neu5Ac, UDP-Gal, UDP-GalNAc, GDP-Fuc, etc.) and D2D1AR2 are the final oligosaccharide compound. Glycosidase reaction can also be performed as an equilibrium reaction. More than one glycosidase and / or transferase can be used for the synthesis of higher oligosaccharides.

Ingående substrat väljs efter vilken oligosackarid som önskas och är ofta lättillgängliga kommersiellt eller syntetiseras med i litteraturen beskriven klassisk organisk-kemisk eller enzymatisk metodik och begränsar ej uppfinningen.Ingredient substrates are selected according to which oligosaccharide is desired and are often readily available commercially or synthesized by classical organic-chemical or enzymatic methodology described in the literature and do not limit the invention.

Enzymer väljs medthänsyn till vilken slutprodukt som önskas. Enzym kan användas in situ (speciellt vissa glykosidaser) eller efter isolering helt eller delvis (speciellt transferaser) från sin naturliga biologiska miljö: Enzymet kan vidare användas i löslig form eller vara immobiliserat till en fast fas genom adsonption, inneslutning, kelering, utfällning, eller kovalent bindning.Enzymes are selected according to the desired end product. Enzyme can be used in situ (especially certain glycosidases) or after isolation in whole or in part (especially transferases) from its natural biological environment: The enzyme can further be used in soluble form or be immobilized to a solid phase by adsorption, entrapment, chelation, precipitation, or covalent bonding.

Samtidig användning av glykosidas och transferas i löslig form eller immobiliseeade till en fast fas (eventuellt samimmobiliserade) är aktuell vid vissa synteser för att omvandla intermediär produktoligosackaridförening- (t.ex. D1AR2 i schemat ovan) så fort den bildas,till färdig produkt (t.ex. D2D1AR2 i schemat). Pâ så sätt vinns en betydande fördel hos sättet enligt uppfinningen eftersom upprening av intermediära produktglykosiden onödiggörs, sekundär hydrolys minimeras (dvs högre utbyte erhålls) och trisackarider eller högre oligosackaridföreningar kan således syntetiseras i ett minimum av "kärl" (i vissa fall enkärlsynteser).Detta möjliggörs av den höga acceptorspecificiteten hos de flesta glykosyltransferaser: trans- feraset i schemat ovan t.ex. reagerar inte med fel isomer av D1AR2 eller med D1R2. Som ett exempel kan nämnas att CMP-N-acetylneuraminat-B-D-galak- tosid (u2-3)sialyltransferas (EC 2.4.99.#) skiljer pà Gal(B1-3)GalNAcR och Gal(B1-3)GlcNAcR som acceptor och att Gal(B1-4)GlcNAcR och GalR är mycket dåliga acceptorer (Sadler et al, J.Biol. Chem. 254, s.4434-4443).Concomitant use of glycosidase and transferase in soluble form or immobilized to a solid phase (possibly co-immobilized) is relevant in some syntheses to convert intermediate product oligosaccharide compound (eg D1AR2 in the scheme above) as soon as it is formed into a finished product (t .ex. D2D1AR2 in the schema). In this way a significant advantage of the method according to the invention is gained because purification of the intermediate product glycoside is unnecessary, secondary hydrolysis is minimized (ie higher yield is obtained) and trisaccharides or higher oligosaccharide compounds can thus be synthesized in a minimum of "vessels" (in some cases monovalent syntheses). is made possible by the high acceptor specificity of most glycosyltransferases: the transferase in the scheme above e.g. does not react with the wrong isomer of D1AR2 or with D1R2. As an example, CMP-N-acetylneuraminate BD-galactoside (u2-3) sialyltransferase (EC 2.4.99. #) Differs between Gal (B1-3) GalNAcR and Gal (B1-3) GlcNAcR as acceptor and that Gal (B1-4) GlcNAcR and GalR are very poor acceptors (Sadler et al., J. Biol. Chem. 254, pp. 444-4443).

Genom att använda glykosider (AR2) som acceptor (AR2 kan syntetiseras enzymatiskt, K.G.I. Nilsson, Carbohydr. Res., (1988) in press) i glykosidas-reaktionen erhålls en slutprodukt som är lätt att isolera pga att ingen anomerisering av produktglykosider sker. Dessutom kan ett och samma glykosidas användas för synthes av flera olika isomerer eftersom regioselektiviteten hos dessa enzymer ofta kan styras genom val av aglykon och konfiguration (d- eller B) på den glykosidiska bindningen 466 405 a, mellan t.ex. A och R2 i schemat ovan (K.G.I. Nilsson, Carbohydr.Res. vol. 167).By using glycosides (AR2) as acceptor (AR2 can be synthesized enzymatically, K.G.I. Nilsson, Carbohydr. Res., (1988) in press) in the glycosidase reaction, a final product is obtained which is easy to isolate due to no anomerization of product glycosides. In addition, one and the same glycosidase can be used for the synthesis of several different isomers since the regioselectivity of these enzymes can often be controlled by the choice of aglycone and configuration (d- or B) on the glycosidic bond 466 405 a, between e.g. A and R2 in the scheme above (K.G.I. Nilsson, Carbohydr.Res. Vol. 167).

R2 kan utgöras av ett organiskt ämne av varierande slag (alifatiskt,aromatiskt etc) som är O-, N-, C-, S-glykosidiskt bundet till A. R2 kan också vara glykosidiskt bundet F. Exempel på organiska ämnen som kan användas är CH3(CH2)n-grupper (metyl, etyl etc), 2-brometyl, allyl, trimethylsilyletyl, 2-(2-karbometoxietyltio)etyl, aminosyror (seryl, treonyl, asparaginyl etc) peptider eller derivat därav, fenyl, bensyl, nitrofenylgrupper, lipid(ana- logaàgrupper.R2 can be an organic substance of various kinds (aliphatic, aromatic, etc.) which is O-, N-, C-, S-glycosidically bound to A. R2 can also be glycosidically bound F. Examples of organic substances that can be used are CH3 (CH2) n groups (methyl, ethyl, etc.), 2-bromomethyl, allyl, trimethylsilylethyl, 2- (2-carbomethoxyethylthio) ethyl, amino acids (seryl, threonyl, asparaginyl, etc.) peptides or derivatives thereof, phenyl, benzyl, nitrophenyl groups , lipid (analog groups).

Exempel på Q- och 5-glykosidaser som kan komma till användning är D-mannosidaser, D-galaktosidaser, L-fukosidaser, N-acetyl-D-galaktosamini- daser, hexosaminidaser och övriga glykosidaser i EC-gruppen 3.2 (se Enzyme Nomenclature (1984) Academic Press). Exempel på lämpliga sialyl- galactosyl-, fukosyl- , N-acetylglucosaminyl-, N-Acetyl-galaktosaminyl, mannosyltransferaser återfinns i Eßegruppen EC 2.4 (Enzyme Nomenclature) Rekombinanta enzymer kan användas enligt uppfinningen.Examples of Q- and 5-glycosidases that can be used are D-mannosidases, D-galactosidases, L-fucosidases, N-acetyl-D-galactosaminidases, hexosaminidases and other glycosidases in the EC group 3.2 (see Enzyme Nomenclature ( 1984) Academic Press). Examples of suitable sialyl-galactosyl-, fucosyl-, N-acetylglucosaminyl-, N-acetyl-galactosaminyl, mannosyltransferases can be found in Eßegruppen EC 2.4 (Enzyme Nomenclature) Recombinant enzymes can be used according to the invention.

Syntesmetoden enligt uppfinningen är generellt tillämpbar för syntes av oligosackaridföreningar som återfinns i glykoproteiner och glykolipider (se referenser på s. 1 ovan). Speciellt intressanta är dessa strukturers minsta fragment som är tillräckliga för att överföra biologisk information och valet av Di och avgörs av detta . Av stort intresse att syntetisera med metoden enligt uppfinningen är blodgruppsdeterminanter, kancerassocierade oligosackaridstrukturer och strukturer med biologisk receptoraktivitet (se referenser på sidan 1 i denna ansökan) Exempel på hur uppfinningen kan komma till praktisk användning beskrivs i nedanstående exempel som dock på intet sätt är avsedda att begränsa uppfinningens omfattning (förkortningarna i denna ansökan för oligosackarider följer IUPAC-IUB:s rekommendationer, J.Biol.Chem., vol. 257,3. 3347-3354 (1982)).The synthetic method of the invention is generally applicable to the synthesis of oligosaccharide compounds found in glycoproteins and glycolipids (see references on page 1 above). Of particular interest are the smallest fragments of these structures that are sufficient to transmit biological information and the choice of Di and are determined by this. Of great interest in synthesizing by the method of the invention are blood group determinants, cancer-associated oligosaccharide structures and structures with biological receptor activity (see references on page 1 of this application). Examples of how the invention may come into practical use are described in the following examples which are in no way intended to limit the scope of the invention (the abbreviations in this application for oligosaccharides follow the recommendations of IUPAC-IUB, J. Biol.Chem., vol. 257.3. 3347-3354 (1982)).

EXEMPEL 1 §y_nt_es_a_v NeESACLQLÉ-Qgal(§_1¿-_3)GalN_a§_(å)¿O§t§r¿ Först syntetiserades Gal(ß1-3)GalNac(ß)-OEtBr. GalNac(ß)~OEtBr erhölls genom att reagera GalNac(ß)-0PhNO2-p (1.2 g) i 100ml natriumfosfat (0.05 M, pH 5.2) med 2-brometanol (10ml) och utnyttjande N-acetyl-3-D-glucosaminidas (EC 3.2.1.30, 70 U). Efter 48 h vid rumstemperatur isolerades 500 mg GalNac(ß)-0EtBr med pelarkromatografi (Kiselgel 60 Merck; metylenklorid- metanol-vatten eluent). GalNAc(ß)-OEtBr (400 må) och Gal(ß)-0PhNO2-o f 466 403 (1 g) suspenderades i 13 ml 0.63 M fosfatbuffert, pH 6.5 och dimethylformamid (4 ml) och 7.2 ml B-D-galaktosidas från oxtestiklar (2 U, Sigma) adderades.EXAMPLE 1 §y_nt_es_a_v NeESACLQLÉ-Qgal (§_1¿-_3) GalN_a§_ (å) ¿O§t§r¿ First Gal (ß1-3) GalNac (ß) -OEtBr was synthesized. GalNac (ß) -OEtBr was obtained by reacting GalNac (ß) -OPHNO2-p (1.2 g) in 100 ml of sodium phosphate (0.05 M, pH 5.2) with 2-bromoethanol (10 ml) using N-acetyl-3-D-glucosaminidase (EC 3.2.1.30, 70 U). After 48 hours at room temperature, 500 mg of GalNac (ß) -OEtBr was isolated by column chromatography (Silica gel 60 Merck; methylene chloride-methanol-water eluent). GalNAc (ß) -OEtBr (400 μl) and Gal (ß) -OPHNO 2 - of 466 403 (1 g) were suspended in 13 ml of 0.63 M phosphate buffer, pH 6.5 and dimethylformamide (4 ml) and 7.2 ml of BD galactosidase from cow sticks ( 2 U, Sigma) was added.

Efter 4 dagar vid 37 OC isolerades produkten med pelarkromatografi (se ovan produktinnehållande fraktioner acetylerades och isolerades med pelarkromato- grafi. Efter deacetylering erhölls 40 mg ren Gal(ß1-3)GalNAc(B)-OEtBr som karakteriserades med NMR. Den så syntetiserade disackaridglykosiden (4 mg) löstes i 2 ml 0.1 M MES-HCl, pH 6.7, tillsammans med 4 mg CMP-Neu5Ac (enzymatiskt preparerad) och 0.17 ml (20mU) av CMP-N-acetylneuraminyl-5-D- galaktosid (G2-3)sialyltransferas (EC 2.4.99.4, submaxillär svinkörtel, Genzyme, Boston) adderades tillsammans med 10fll Triton-X-100 and 2 mg bovint serum albmin. Mer CMP-Neu5Ac (4 mg)adderades efter 20 H. Efter tøtait 3 dygn vid 37 Oc renaaes 5 mg NeuSAC(az-s)Ga1 fram mha pelarkromatografi (Kiselgel 60, acetonitril-isopropanol-2.5M NH4OH och Sephadex GF15). Produkten var ren enligt NMR (1H, 130). Metylerings- analys tydde på rätt produkt.After 4 days at 37 ° C, the product was isolated by column chromatography (see above product-containing fractions were acetylated and isolated by column chromatography. After deacetylation, 40 mg of pure Gal (ß1-3) GalNAc (B) -OEtBr was obtained which was characterized by NMR. The disaccharide glycoside thus synthesized (4 mg) was dissolved in 2 ml of 0.1 M MES-HCl, pH 6.7, together with 4 mg of CMP-Neu5Ac (enzymatically prepared) and 0.17 ml (20mU) of CMP-N-acetylneuraminyl-5-D-galactoside (G2-3 ) sialyltransferase (EC 2.4.99.4, submaxillary swine gland, Genzyme, Boston) was added together with 10fl L Triton-X-100 and 2 mg bovine serum albumin More CMP-Neu5Ac (4 mg) was added after 20 H. After tøtait 3 days at 37 Oc purified 5 mg NeuSAC (az-s) Ga1 obtained by column chromatography (Silica gel 60, acetonitrile-isopropanol-2.5M NH 4 OH and Sephadex GF15) The product was pure by NMR (1H, 130) Methylation analysis indicated the correct product.

EXEMPEL 2 Syntes av Neu5Ac(a2-3)Gal(ß1-3)GlcNAc(ß)-OMe: Denna substans preparerades 'analogt. g-D-Galaktosid (a2-3)sialyltransferas (0.34 ml, 40 mU) adderades till 0.7 ml O.1M MES-HCl, pH 6.7, som innehöll 30 mg Gal(ß1-3)GlcNAc(ß)-OMe (syntes analog med ovan med GlcNAc(B)-OMe som acceptor), 10 mg CMP-Neu5Ac, 5 H1 Triton-X-100 och 1 mg albumin. Mer CMP-Neu5Ac (10 mg) adderades efter 30 h. Efter totalt 5 dygn vid 37 OC renades produkten med pelarkromatografi pålkiselgel 60 och på Sephadex G-15 och 10 mg Neu5Ac(G2-3)Gal(31-3)GlcNAc(ß)-OMe erhölls. Produkten var ren enligt NMR och metyàèringsanalys och elementar analys pekade på rätt produkt.EXAMPLE 2 Synthesis of Neu5Ac (α2-3) Gal (β1-3) GlcNAc (β) -OMe: This substance was prepared analogously. β-Galactoside (α2-3) sialyltransferase (0.34 ml, 40 mU) was added to 0.7 ml O.1M MES-HCl, pH 6.7, which contained 30 mg of Gal (ß1-3) GlcNAc (ß) -OMe (synthesis analogous to above with GlcNAc (B) -OMe as acceptor), 10 mg CMP-Neu5Ac, 5 H1 Triton-X-100 and 1 mg albumin. More CMP-Neu5Ac (10 mg) was added after 30 hours. After a total of 5 days at 37 ° C, the product was purified by column chromatography on silica gel 60 and on Sephadex G-15 and 10 mg Neu5Ac (G2-3) Gal (31-3) GlcNAc (ß ) -OMe obtained. The product was pure according to NMR and methylation analysis and elemental analysis pointed to the correct product.

Claims (9)

466 405 PATENTKRAV466 405 PATENT REQUIREMENTS 1. Sätt att från minst en oligosackarid eller minst en monosackaridglykosid, minst ett nukleotidsocker (donatorsubstanser) och minst en acceptorsubstans, syntetisera en oligosackaridförening, som antingen utgör eller är ett fragment eller en analog av kolhydratdelen i ett glykokonjugat, kännetecknat av att minst ett glykosidas (EC- klass 3.2) används för syntes via omvänd hydrolys eller transglykosylering och minst ett glykosyltransferas (EC-klass 2.4) används för syntesen, samt att den önskade oligosackaridföreningen isoleras från reaktionsblandningen.A method of synthesizing from at least one oligosaccharide or at least one monosaccharide glycoside, at least one nucleotide sugar (donor substances) and at least one acceptor substance, an oligosaccharide compound which either constitutes or is a fragment or an analogue of the carbohydrate moiety of a glycoconjugate (EC class 3.2) is used for synthesis via reverse hydrolysis or transglycosylation and at least one glycosyltransferase (EC class 2.4) is used for the synthesis, and the desired oligosaccharide compound is isolated from the reaction mixture. 2. Sätt enligt krav 1, kännetecknat av att i donator och acceptorsubstansernas kolhydratdel ingår en eller flera av monosackariderna D-galaktos, D-mannos, N-acetylneuraminsyra, N-acetyl-D-galaktosamin, N-acetyl-D-glukosamin och L-fukos eller en analog av någon av dessa.Process according to Claim 1, characterized in that the carbohydrate moiety of the donor and the acceptor substances includes one or more of the monosaccharides D-galactose, D-mannose, N-acetylneuraminic acid, N-acetyl-D-galactosamine, N-acetyl-D-glucosamine and L -fucose or an analogue of any of these. 3. Sätt enligt krav 1-2, kännetecknat av att acceptorsubstansen är en glykosid vars aglykon utgörs av glykosidiskt bundet fluor eller ett O-, N-, C-, eller S- glykosidiskt bundet alifatiskt eller aromatiskt ämne.Process according to Claims 1 to 2, characterized in that the acceptor substance is a glycoside whose aglycone consists of glycosidically bound fluorine or an O-, N-, C- or S-glycosidically bound aliphatic or aromatic substance. 4. Sätt enligt krav 3, kännetecknat av att det alifatiska eller aromatiska ämnet är lämpligt för temporärt skydd av anomert kol, för polymerisation, för konstruktion av affinitetsmarkörer till proteiner, för konstruktion av spacerglykosid, för koppling till aminosyra, peptid, lipid(analog) eller affinitetsbärare, eller är en aminosyra, peptid, lipid(analog) eller är ett ämne som möjliggör användning av oligosackaridföreneningen som inhibitor.A method according to claim 3, characterized in that the aliphatic or aromatic substance is suitable for temporary protection of anomeric carbon, for polymerization, for construction of affinity markers for proteins, for construction of spacer glycoside, for coupling to amino acid, peptide, lipid (analogue) or affinity carrier, or is an amino acid, peptide, lipid (analog) or is a substance that enables the use of the oligosaccharide compound as an inhibitor. 5. Sätt enligt krav 3 eller 4, kännetecknat av att det alifatiska eller aromatiska ämnet utgörs av en allyl-, metyl-, etyl-, bromoetyl-, epoxi-, trimetylsilyletyl-, fenyl-, bensyl-, eller en nitrofenylgrupp.Process according to Claim 3 or 4, characterized in that the aliphatic or aromatic substance consists of an allyl, methyl, ethyl, bromoethyl, epoxy, trimethylsilylethyl, phenyl, benzyl or nitrophenyl group. 6. Sätt enligt krav 1-5, kännetecknat av att nukleotidsockret utgörs av något eller några av CMP-Neu5Ac, GDP-Fuc, UDP-Gal,Method according to claims 1-5, characterized in that the nucleotide sugar consists of one or more of CMP-Neu5Ac, GDP-Fuc, UDP-Gal, 7. UDP-GICNAC, UDP-GâlNAC. n 7. Sätt enligt något eller flera av kraven 1-6, kännetecknat av att minst ett glykosidas och minst ett glykosyltransferas används samtidigt för syntesen, antingen i löslig form eller separat- eller samimmobiliserade till en fast fas.7. UDP-GICNAC, UDP-GâlNAC. n Process according to one or more of Claims 1 to 6, characterized in that at least one glycosidase and at least one glycosyltransferase are used simultaneously for the synthesis, either in soluble form or separately or co-immobilized to a solid phase. 8. Sätt enligt något eller flera av kraven 1-7, kännetecknat av att Neu5Acd2-3Ga1ß1-3GlcNAcR eller Neu5Acü2-3Galß1-3GalNAcR syntetiseras genom att använda B-D-galaktosidas och ß-D-galaktosid «x2-3)sialyltransferas och där R är en glykosidiskt bunden aglykon. 466 403Method according to one or more of Claims 1 to 7, characterized in that Neu5Acd2-3Ga1ß1-3GlcNAcR or Neu5Acü2-3Galß1-3GalNAcR is synthesized using BD-galactosidase and β-D-galactoside ( a glycosidically bound aglycone. 466 403 9. Sätt enligt ett eller flera av de föregående kraven, kännetecknat av att en oligosackaridförening med biospecifik affinitet till ett annat ämne syntetiseras och isoleras.Method according to one or more of the preceding claims, characterized in that an oligosaccharide compound with biospecific affinity for another substance is synthesized and isolated.
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DE68923177T DE68923177T2 (en) 1988-03-24 1989-03-22 METHOD FOR PRODUCING OLIGOSACCHARIDES.
US07/603,699 US5246840A (en) 1988-03-24 1989-03-22 Method for synthesis of oligosaccharides
PCT/SE1989/000151 WO1989009275A1 (en) 1988-03-24 1989-03-22 A method for synthesis of oligosaccharides
EP89904653A EP0432157B1 (en) 1988-03-24 1989-03-22 A method for synthesis of oligosaccharides

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WO1989009275A1 (en) 1989-10-05
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SE8801080D0 (en) 1988-03-24
DE68923177T2 (en) 1996-02-15
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SE8801080L (en) 1989-09-25

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